There have been various cytokines identified that inhibit manifestations of biological functions such as immunoresponse, inflammatory reactions, and hemopoietic functions in the body. The structures and activities of these molecules have gradually been clarified. With such clarification, it has also been made clear that these cytokine molecules are effective not only on the immune system, but also on various other biological functions.
In fact, a large number of human diseases involve TNF molecules including endotoxic shock, inflammation, hemorrhagic necrosis of tumors, cytotoxicity, (reviewed Tracey, K. and Cerami, A. (1994), Annu Rev Med 45: 491-503, Vassalli, P., (1992) Annu Rev Immunol 10:411-452), and obesity-linked insulin resistance (Hotamisligil, G. et al., Science 259:87-91), to name a few. The TNF superfamily currently includes 17 members in vertebrates (review Locksley, R. et al. (2001) Cell 104: 487-501). This family exhibits the highest homology between their C-terminal, receptor binding domains. The superfamily members are type II membrane proteins that act in an autocrine, paracrine, or endocrine manner either as integral membrane proteins or as proteolytically processed soluble factors (Banner et al. (1993), Cell 73:431-445).
Most members within this ligand family are expressed in the immune system and play important roles in immune system development and modulation (Smith et al., (1994) Cell 76:959-962). For example, TNFalpha is expressed in macrophages and is a critical mediator of inflammatory responses and immune defenses (Tracey and Cerami, 1994). Pharmaceuticals to inhibit TNF control pathogenic inflammatory responses such as rheumatoid arthritis and inflammatory bowel disease (Maini, R. and Taylor, P. (2000) Annu. Rev., Med, 51, 207-229, Papadakis, K. and Targan, S. (2000) Annu. Rev. Med. 51, 289-298.)
Despite the functional redundancy of this family, specificity may be accomplished by coordinating the spatial and temporal expression of TNF-related ligands and their receptors, and by restricting the expression of signal transduction molecules to specific cell types. Many TNF receptors have been identified and share characteristic multiple cysteine repeats within their extracellular domain and do not possess cytoplasmic catalytic domains. TNF receptors interact with a family of molecules called TRAFs (TNF receptor associated proteins) that act as adaptors for downstream signaling events. Hence, binding of a TNF cytokine to its cognate receptor, which is interacting with TRAF, leads to the activation of several signal transduction pathways, including the activation of the cascade of caspase/ICE-like proteases, which are responsible for apoptosis. Also activated is the Re1 protein family of transcription factors, which inhibit apoptosis, and mitogen activated protein kinases including the Jun N-terminal protein kinases (JNK) and the extracellularly-regulated kinases (ERK) (Locksley et al., 2001).
Among the cytokines, TNF was identified as an antitumor cytokine and has been expected to be useful as an antitumor agent. However, later it was reported that TNF has an activity of stimulating production of other cytokines such as IL-1, etc., proliferative activity of fibroblasts, endotoxin shock-inducing activity, an activity of promoting the adhesion of leukocytes to endothelium by increasing intercellular adhesion molecules (ICAM-1, ICAM-2) or endothelial leukocyte adhesion molecule-1 (ELAM-1), an activity of bone absorption, and an activity of inducing arthritis (e.g. cartilage decomposing activity). (Beutler, B., et al., Nature, 316, 552-554 (1985); Peetre, C., et al., J. Clin. Invest., 78, 1694-1700 (1986); Kurt-Jones, E. A., et al., J. Immunol., 139, 2317-2324 (1987); Bevilacqua, M. P., et al., Science, 243, 1160-1165 (1989); Akatu, K. & Suda, T., Medical Practice, 8 (9), 1393-1396 (1991).
Moreover, it is reported that in bacterial or parasitic infectious diseases, TNF is contained in a higher concentration in blood and cerebrospinal fluid (M1 tsuyama, M., IGAKU-NO-AYUMI, 159 (8), 467-470 (1991); and Masayasu, N., IGAKU-NO-AYUMI, 159 (8), 471-474 (1991)). It is also reported that in rheumatoid arthritis, the joint fluid and blood serum have TNFα activity. (Saxne, T., et al., Arthritis Rheum., 31, 1041 (1988); Chu, C. Q., et al., Arthritis Rheum., 34, 1125-1132 (1991); Macnaul, K. L., et al., J. Immunol., 145, 4154-4166 (1990); Brennan, F, M., et al., Eur, J. Immunol., 22(7):1907-12, (1992); and Brennan, F. M., et al., Bri. J. Rheum., 31, 293-298 (1992)).
It is further reported that in patients suffering from adult respiratory distress syndrome (ARDS), the phlegm of the patients contain increased levels of TNF (Millar, A. B., et al., Nature, 324, 73 (1986)), and that TNF also participates in the severity of virus hepatitis. (Muto, Y., et al., Lancet, 2(8602):72-4, (1988)).
It is also reported that the blood concentration of TNFα rises in cases of myocardial ischemia (e.g. acute myocardial infarction) (Latini, R., et al., J. Cardiovasc. Pharmacol., 23, 1-6 (1994)), and it is suggested that TNFα is involved in such diseases. (Lefer, A. M., et al., Science, 249, 61-64 (1990)). It has also been reported that TNFα inhibits myocardial contraction. (Finkel, M. S., et al., Science, 257, 387-389 (1992); and Pagani, D. F., et al., J. Clin. Invest., 90, 389-398 (1992)).
No known invertebrate TNF molecules have been reported to date, although many TNF intracellular signaling molecules such as TRAFs and Re1 proteins have been identified in Drosophila melagaster (Khush, R. and Lemaitre, B. (2000) Trends Genet. 16:442-449). Identification of a novel TNF in Drosophila provides, for the first time, a useful tool for genetic and molecular study of TNF biology and the subsequent validation of such molecules as pharmaceutical targets. The present invention provides the encoding polynucleotide and polypeptide sequence of a novel Drosophila TNF protein. The present invention also provides methods linking the inventive DmTNF to the Re1 protein signal transduction in Drosophila. The use of Drosophila as a model organism with genetic and other technologies would be useful in the elucidation of biological pathways, particularly those related to TNF (Margolis, J. and Duyk, G. Nat. Biotechnol. (1998)16: 311, Matthews D. and Kopczynski, J (2001) Drug Disc., Today 6: 141-149).
Therefore, the development of therapeutics that modulate (i.e., act as antagonists or agonists of TNF) is important to treat diseases related to TNF.